CN109381429B - Paclitaxel targeted sustained-release liposome modified by leukocyte membrane and preparation method thereof - Google Patents
Paclitaxel targeted sustained-release liposome modified by leukocyte membrane and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a paclitaxel targeted sustained-release liposome modified by a leukocyte membrane and a preparation method thereof, belonging to the technical field of controlled release of drugs and biological materials. The paclitaxel targeted sustained-release liposome comprises a drug-loaded sustained-release liposome composed of paclitaxel, star-shaped cholic acid functionalized polylactic acid, soybean phospholipid and cholesterol, and a leukocyte membrane for modifying the surface of the drug-loaded sustained-release liposome. The star cholic acid functionalized polylactic acid with excellent biocompatibility is introduced into the paclitaxel sustained-release liposome, and the surface modification is carried out by using a white cell membrane, so that the prepared paclitaxel targeted sustained-release liposome overcomes the toxic and side effects brought by the surfactant polyoxyethylene castor oil in the current paclitaxel injection, and has the advantages of excellent sustained-release effect, better tumor targeting property, long in-vivo half-life period, high stability, good biocompatibility and the like.
Description
Technical Field
The invention belongs to the technical field of drug controlled release and biological materials, and particularly relates to a paclitaxel targeted sustained-release liposome modified by a leukocyte membrane and a preparation method thereof.
Background
Paclitaxel is a diterpenoid compound extracted from Taxus plants of Taxaceae, has unique anti-tumor effect, can form stable microtubule bundle after being combined with tubulin, and can inhibit the replication of cancer cells, thereby preventing the proliferation of cancer cells, belongs to broad-spectrum and high-efficiency anticancer drugs, and is mainly used for treating cancers such as ovarian cancer, breast cancer, melanoma, lung cancer, carcinoma of large intestine, head and neck cancer, lymphoma, cerebroma, etc.
However, due to the special physicochemical properties and pharmacological actions of paclitaxel, the clinical use still has the disadvantages of non-ideal curative effect and obvious toxic and side effects, and the main problems are as follows: firstly, the solubility in water is poor, a surfactant polyoxyethylene castor oil is added into a commercial paclitaxel injection, degradation products of the polyoxyethylene castor oil in vivo easily cause severe adverse reactions such as anaphylactic reaction, neurotoxicity, nephrotoxicity and the like, and dexamethasone is required to be given as a preventive medicine before the medicine is taken in order to overcome the anaphylactic reaction; paclitaxel is used as a cell cycle specific drug, the tumor inhibition effect of low-dose continuous administration in vivo is more obvious than that of one-time impact administration, and the paclitaxel is supposed to be prepared into a sustained-release preparation, but the paclitaxel injection sold in the market at present is a non-sustained-release preparation, so that the exertion of the curative effect is influenced; the targeted preparation can distribute the medicine to the target organ to the maximum extent to achieve the best curative effect, but has no effect on other normal tissues and organs, thereby achieving the high-efficiency and low-toxicity treatment effect, being particularly suitable for anti-cancer medicines, and the paclitaxel injection sold in the market at present is a non-targeted preparation, which influences the clinical application of the paclitaxel injection.
Therefore, finding new formulations aiming at improving water solubility, delaying drug release and increasing targeting effect becomes a hotspot of paclitaxel drug research, for example, paclitaxel is prepared into new formulations such as liposome, nanoparticle, emulsion, clathrate, prodrug, micelle, and the like, wherein the liposome is widely applied to paclitaxel carriers because of the advantages of excellent biocompatibility, simple preparation process, capability of simultaneously loading hydrophilic and hydrophobic drugs, and the like, but the liposomes still have some disadvantages and problems to be solved urgently: firstly, the traditional unmodified common liposome is easily damaged by various proteins and enzymes in blood, for example, high-density lipoprotein is a main component for damaging the liposome, apolipoprotein is easily shed from the high-density lipoprotein and combined with phospholipid in the liposome, and exchange with the phospholipid is easily generated, so that the liposome forms a pore to form a hole and leak medicines; the complex formed by combining the serum albumin and the liposome phospholipid causes the reduction of the liposome stability; the liposome can activate a complement system in blood to form a complex for destroying membranes, so that hydrophilic channels appear on the liposome membranes to cause drug leakage, and a large amount of water and electrolyte enter, even the liposome can be cracked; phospholipase in blood can hydrolyze phospholipid in liposome, resulting in liposome destruction (the reaction strength is determined by phospholipid structure); secondly, after the liposome enters a blood circulation system, most unmodified liposome is transported to parts rich in mononuclear phagocyte systems such as liver, spleen and the like, a small amount of unmodified liposome is absorbed by lung, bone marrow and kidney, and a hepatocyte membrane receptor identifies a phospholipid negative electricity radical directly exposed on the surface, so that the phospholipid negative electricity radical is phagocytized by hepatocyte, and the retention time of the common liposome in systemic circulation is too short; the unmodified traditional liposome has an unsatisfactory slow release effect due to the easiness of destroying the phospholipid, and the clinical curative effect is influenced; the unmodified traditional liposome has unsatisfactory targeting property and great toxic and side effects, and the clinical application of the unmodified traditional liposome is seriously influenced.
Therefore, the development of the paclitaxel targeted sustained-release liposome which has superior sustained-release effect, high stability and good targeting property and avoids the damage of various factors in vivo has important clinical significance.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides the paclitaxel targeted sustained-release liposome modified by the leucocyte membrane, which has the advantages of targeting effect and capability of slowly releasing the drug in vivo, thereby prolonging the half-life period of the drug, reducing the administration times, improving the curative effect and reducing the toxic and side effects.
The invention provides a paclitaxel targeted sustained-release liposome modified by a leukocyte membrane, which comprises a drug-loading sustained-release liposome composed of paclitaxel, star-shaped cholic acid functionalized polylactic acid, soybean phospholipid and cholesterol and a leukocyte membrane modified on the surface of the drug-loading sustained-release liposome, and the specific mass percentages are as follows: 5-10% of paclitaxel, 50-70% of soybean phospholipid, 15-25% of cholesterol, 6-12% of star-shaped cholic acid functionalized polylactic acid and 2-6% of leukocyte membrane.
The invention further provides a preparation method of the paclitaxel targeted sustained-release liposome modified by the leucocyte membrane, which comprises the following steps:
(1) preparing paclitaxel sustained-release liposome: weighing paclitaxel, soybean phospholipid, cholesterol and star-shaped cholic acid functionalized polylactic acid, dissolving in chloroform, performing reduced pressure rotary evaporation at 35-40 ℃ to remove the solvent until a layer of liposome film is formed on the inner wall of the eggplant-shaped bottle, placing in a vacuum drier for 24h, adding phosphate buffer (pH7.4), performing rotary hydration in a constant-temperature water bath at 40 ℃ for 1h, performing ultrasonic treatment on an ice bath probe for 4-6 min, treating by a micro-jet high-pressure homogenizer (the homogenizing pressure is 120MPa, the homogenizing temperature is 37 ℃, the homogenizing times are 5 times), and filtering by a 0.22 mu m microporous membrane to obtain a paclitaxel sustained-release liposome suspension;
(2) preparation of leukocyte membrane: separating leucocytes: selecting rats with the age of 3-5 weeks, collecting whole blood through eye sockets, diluting the whole blood with an equivalent amount of EDTA-PBS buffer solution (0.2mol/L, pH7.4), and adding the diluted whole blood into a centrifuge tube for later use; mixing 9% of ficoll and 15.2% of diatrizoate meglumine, preparing a solution with the specific gravity of 1.114g/ml by using distilled water, adjusting the osmotic pressure to 540mOsm/L by using a 0.3% sodium chloride solution, adding the solution into the centrifugal tube along the tube wall, centrifuging for 30min by using the centrifugal force of 700 Xg, slowly sucking a middle milky cloudy layer (leucocyte layer) by using a liquid transfer gun, washing for 2 times by using EDTA-PBS buffer solution, centrifuging, and collecting leucocytes;
separating and purifying the leucocyte membrane: collected leukocyte hypotonic lysis solution (1mM NaCl, 1mM MgCl) for leukocytes25ug/ml RNase, 8 ug/ml DNase), homogenizing with a hand-held Dounce homogenizer for 2min in ice bath, performing hypotonic lysis at 4 ℃ for 24h, centrifuging at a centrifugal force of 1200 Xg for 10min, discarding the supernatant, centrifuging the collected leukocyte membrane with gradient sucrose solutions of 30%, 45% and 50% at a centrifugal force of 24000 Xg at 4 ℃ for 30min, collecting the purified leukocyte membrane, and placing in a refrigerator at 2-6 ℃ for later use;
(3) modification of paclitaxel sustained-release liposome by leukocyte membrane: and oscillating and mixing the prepared leucocyte membrane and the liposome, incubating and fusing for 12h, centrifuging, washing twice by using PBS buffer solution, resuspending by using distilled water, performing ultrasonic treatment for 60s by using an ultrasonic instrument at the frequency of 35kHz and the power of 80W, passing through a 0.22 mu m microporous filter membrane, performing high-speed centrifugation, discarding supernatant, and performing freeze drying to obtain the taxol targeted sustained-release liposome modified by the leucocyte membrane.
Compared with the prior art, the invention has the advantages and innovation points that:
(1) the paclitaxel is a fat-soluble drug, is wrapped between lipid bilayers in the liposome, and the addition of the star-shaped cholic acid functionalized polylactic acid can simultaneously increase the drug-loading rate and the stability of the paclitaxel in the preparation and can play a role in delaying the release of the drug. The traditional liposome which is composed of lecithin and cholesterol only has poor stability, the liposome membrane is easy to form holes, the medicine leaks, water and electrolyte enter in a large amount, and even the osmotic lysis of the liposome is caused. The introduction of the star-shaped cholic acid functionalized polylactic acid is embedded between the lipid bilayers due to the special three-dimensional structure, so that the lipid bilayers are more compact and stable, the generation of holes is blocked, the too fast release of the medicine is prevented, the slow and continuous low-dose medicine release is achieved, the half life of the medicine is prolonged, and the paclitaxel is used as a medicine with cell cycle specificity, and the sustained release preparation can obviously improve the curative effect and reduce the toxic and side effects.
(2) The leucocyte membrane is prepared and modified on the surface of the paclitaxel slow release liposome by collecting whole blood and separating leucocytes, and the leucocyte modified liposome has the active targeting effect because the leucocyte modified liposome can identify and gather in tumor cells, can improve the curative effect of paclitaxel and reduces the toxic and side effects because the leucocyte modified liposome is less transported to normal tissues and organs. Numerous studies have also shown that leukocyte membrane-modified drug delivery systems prevent the binding of many different components of the blood, particularly opsonins, to them, thereby reducing the affinity to the mononuclear phagocyte system, reducing the phagocytosis or uptake by the reticuloendophagous system, stabilizing the circulation, significantly prolonging half-life, and enhancing the therapeutic effect of the drug.
(3) The paclitaxel sustained-release liposome is prepared by combining a film dispersion method and a high-pressure homogenization process for the first time, and the liposome preparation with small particle size (within 100nm, nano preparation, injectable administration), small polydispersion coefficient (uniform particle size) and good stability can be obtained.
Drawings
FIG. 1 is the in vitro cumulative drug release percentage of the paclitaxel targeted sustained release liposome prepared in example 1.
FIG. 2 is the in vitro cumulative drug release percentage of the paclitaxel targeted sustained release liposome prepared in example 2.
FIG. 3 is the in vitro cumulative drug release percentage of the paclitaxel targeted sustained release liposome prepared in example 3.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes and modifications can be made by one skilled in the art after reading the teachings of the present invention, and equivalents fall within the scope of the invention defined by the appended claims.
Example 1
1. A preparation method of paclitaxel targeting sustained-release liposome modified by leucocyte membranes comprises the following steps:
(1) preparing paclitaxel sustained-release liposome: weighing 5mg of paclitaxel, 65mg of soybean phospholipid, 16mg of cholesterol and 10mg of star-shaped cholic acid functionalized polylactic acid, dissolving in a certain amount of chloroform, performing reduced pressure rotary evaporation at 37 ℃ to remove the solvent until a layer of liposome film is formed on the inner wall of the eggplant-shaped bottle, placing in a vacuum drier for 24h, adding a phosphate buffer solution (pH7.4), performing rotary hydration in a constant-temperature water bath at 40 ℃ for 1h, performing ultrasonic treatment on an ice bath probe for 4-6 min, processing by a micro-jet high-pressure homogenizer (the homogenizing pressure is 120MPa, the homogenizing temperature is 37 ℃, the homogenizing times are 5 times), and passing through a 0.22 mu m microporous filter membrane to obtain a paclitaxel sustained-release liposome suspension;
(2) preparation of leukocyte membrane: separating leucocytes: selecting rats with the age of 3-5 weeks, collecting whole blood through eye sockets, diluting the whole blood with an equivalent amount of EDTA-PBS buffer solution (0.2mol/L, pH7.4), and adding the diluted whole blood into a centrifuge tube for later use; mixing 9% of ficoll and 15.2% of diatrizoate meglumine, preparing a solution with the specific gravity of 1.114g/ml by using distilled water, adjusting the osmotic pressure to 540mOsm/L by using a 0.3% sodium chloride solution, adding the solution into the centrifugal tube along the tube wall, centrifuging for 30min by using the centrifugal force of 700 Xg, slowly sucking a middle milky cloudy layer (leucocyte layer) by using a liquid transfer gun, washing for 2 times by using EDTA-PBS buffer solution, centrifuging, and collecting leucocytes;
separating and purifying the leucocyte membrane: collected leukocyte hypotonic lysis solution (1mM NaCl, 1mM MgCl) for leukocytes25ug/ml RNase, 8 ug/ml DNase), homogenizing in ice bath for 2min with a handheld Dounce, performing hypotonic lysis at 4 ℃ for 24h, centrifuging at a centrifugal force of 1200 Xg for 10min, discarding the supernatant, centrifuging the collected leukocyte membrane at a centrifugal force of 24000 Xg for 30min at 4 ℃ with gradient sucrose solutions of 30%, 45% and 50% respectively, collecting the purified leukocyte membrane, and placing in a refrigerator at 2-6 ℃ for later use.
(3) Modification of paclitaxel sustained-release liposome by leukocyte membrane: shaking and mixing the prepared leucocyte membrane (4mg) and liposome, incubating and fusing for 12h, centrifuging, washing twice with PBS buffer solution, resuspending with distilled water, using Fisher scientific FS30D ultrasonic instrument, performing ultrasonic treatment for 60s at the frequency of 35kHz and the power of 80W, passing through a 0.22 mu m microporous filter membrane, centrifuging at high speed, discarding supernatant, and freeze-drying to obtain the paclitaxel targeted sustained-release liposome modified by the leucocyte membrane.
2. Pharmaceutics characterization of paclitaxel targeted sustained-release liposome
(1) Particle size and polydispersity: the particle size is measured by a laser scattering particle size distribution instrument, and the measurement result is as follows: the average grain diameter is 80.1 nm; the polydispersity was 0.171.
(2) And surface appearance: dispersing paclitaxel targeted sustained-release liposome in distilled water, dripping on copper mesh coated with Fomva membrane, drying, dripping 2% phosphotungstic acid for negative staining, naturally drying, and observing the shape and structure of the microparticle with a transmission electron microscope (TecnaiG20 transmission electron microscope). As a result: the surface appearance is spherical, and the size distribution is relatively uniform.
(3) And the determination and the result of the encapsulation efficiency are as follows:
demulsifying the prepared paclitaxel target sustained-release liposome with methanol, and measuring paclitaxel content by HPLC to obtain encapsulated and unencapsulated paclitaxel total amount, which is recorded as rhoGeneral assembly(ii) a Mixing paclitaxel targeting sustained release liposomeCentrifuging the suspension at high speed (15000r/min) for 30min, collecting supernatant, demulsifying with methanol, and measuring paclitaxel content by HPLC to obtain encapsulated paclitaxel content, which is recorded as ρBag (bag). Wherein the chromatographic conditions are as follows: the mobile phase is methanol-water (70: 30); the chromatographic column is elette C18(250mm × 4.6mm, 5 μm); the detection wavelength is 227 nm; the flow rate is 1.0 ml/min; the column temperature was 25 ℃; the amount of sample was 20. mu.L. According to the relevant regulations in Chinese pharmacopoeia 2015 edition, the liposome encapsulation efficiency is the encapsulated drug amount in the system/the total drug amount encapsulated and unencapsulated in the system x 100%. And (3) measuring results: the encapsulation efficiency was 83.6%.
3. In vitro cumulative drug release percentage test
Weighing 6mg of paclitaxel targeted sustained-release liposome, adding into 6mL of phosphate buffer (pH7.4), ultrasonically dispersing, filling into a dialysis bag (model 4000), placing into a 30mL centrifuge tube, shaking the external liquid in a 1% DMSO aqueous solution in a shaking table at 37 ℃, taking out all the external liquid at a preset time point, standing for 6h, measuring the paclitaxel concentration by using an ultraviolet spectrophotometer ((Perkin-Elmer Lambda Bio 40UV/VIS detector)), measuring the wavelength at 227nm, and calculating the cumulative drug release percentage according to the following formula:
cumulative percent (%) released is the amount of drug released/total amount of drug entrapped x 100%
The paclitaxel targeted liposome without adding the star-shaped cholic acid functionalized polylactic acid (other components and processes are the same) is used as a control preparation, and the cumulative drug release percentage is determined and calculated according to the same in vitro cumulative drug release percentage test method.
The result is shown in fig. 1 in detail, compared with the paclitaxel targeted liposome without the star-shaped cholic acid functionalized polylactic acid, the paclitaxel targeted sustained-release liposome prepared in this example releases the drug more slowly, the cumulative drug release percentages of 1, 2, 3, 6, 9 and 12h are respectively 6.4, 15.1, 22.3, 43.9, 55.4 and 63.3, and the cumulative drug release percentages of the paclitaxel targeted liposome without the star-shaped cholic acid functionalized polylactic acid at the corresponding time points are respectively 31.5, 45, 56.3, 68.2, 75.4 and 79.8, which indicates that the addition of the star-shaped cholic acid functionalized polylactic acid can play a role in delaying the drug release. The traditional liposome which is composed of lecithin and cholesterol only has poor stability, the liposome membrane is easy to form holes, the medicine leaks, water and electrolyte enter in a large amount, and even the osmotic lysis of the liposome is caused. The introduction of the star-shaped cholic acid functionalized polylactic acid is embedded between lipid bilayers due to a special three-dimensional structure, so that the lipid bilayers are more compact and stable, the generation of holes is blocked, the too fast release of the medicine is prevented, the slow and continuous low-dose medicine release is achieved, the half-life period of the medicine is prolonged, the effective medicine concentration is maintained for a longer time, and the taxol is used as a medicine with cell cycle specificity, and the sustained-release preparation can continuously and effectively inhibit the growth of cancer cells, remarkably improve the curative effect, reduce the toxic and side effects and improve the medication compliance of patients.
4. Pharmacokinetics and tissue distribution in vivo
4.1 establishing a nude mouse breast cancer animal model: culturing MCF-7 cells of human breast cancer to logarithmic growth phase, counting with cell counting plate, and adjusting cell concentration to 1 × 107Taking 0.1ml, inoculating under the second pair of mammary glands pads on the right side of SPF-grade BALB/c female nude mice, and aseptically feeding until the tumor volume is 100-200 mm3。
4.2 dosing and sampling: randomly dividing tumor-bearing nude mice into 3 groups, respectively injecting taxol (paclitaxel injection) into tail vein of single dose, paclitaxel targeted liposome without adding star-shaped cholic acid functionalized polylactic acid and paclitaxel targeted sustained-release liposome (paclitaxel dosage is 10mg/kg), respectively removing eyeball and blood after injection for 15min, 30min, 1h, 3h, 6h, 9h, 12h, 18h and 24h, centrifuging after anticoagulation treatment and taking blood plasma, storing at-80 ℃, and taking heart, liver, spleen, lung, kidney and tumor tissues after nude mice at 1h, 6h, 12h and 24h are killed by cervical dislocation method, cleaning, weighing, and storing at-80 ℃ for later use.
4.3 blood concentration determination and pharmacokinetic parameters: standing the plasma sample to room temperature, adding anhydrous methanol to fully precipitate protein, centrifuging, collecting supernatant, vacuum rotary evaporating, dissolving residue with mobile phase, and determining paclitaxel content by high performance liquid chromatography, wherein the chromatographic conditions are as follows: the mobile phase is methanol-water (70: 30); the chromatographic column is elette C18(250mm × 4.6mm, 5 μm); the detection wavelength is 227 nm; the flow rate is 1.0 ml/min; column temperature of25 ℃; the amount of sample was 20. mu.L. The blood drug concentration at each time point was input into the pharmacokinetic 3P87 program to have a degree of fitting γ2The atrioventricular model was determined for the index and pharmacokinetic parameters were output with the results detailed in table 1.
TABLE 1 in vivo pharmacokinetic parameters of different formulations of paclitaxel (example 1)
Tail vein injection of taxol (paclitaxel injection) for group "1" in the table; group 2' tail vein injection paclitaxel targeting liposome without star cholic acid functionalized polylactic acid; group 3 tail vein injection paclitaxel targeting slow release liposome.
And (3) analyzing pharmacokinetic parameters: model chimeric shows that the in vivo process of different formulations of paclitaxel is in a two-chamber model, and the half-life period t of taxol injection1/2AUC of area under the drug-time curve0-∞The mean residence time MRT is smaller than that of the other two groups, and the clearance rate CL is the largest, which shows that the commercial paclitaxel common injection liquid has high elimination speed in vivo, short mean residence time in vivo and needs frequent administration, after the paclitaxel common injection liquid is prepared into a liposome preparation, the half life is prolonged, the area under the curve and the mean residence time are increased, because the liposome releases the medicament slowly, and the release of the paclitaxel can be further slowed down after the liposome is modified by a leukocyte membrane. In addition, numerous studies have shown that leukocyte membrane-modified drug delivery systems prevent binding of many different components of the blood, particularly opsonins, to them, thereby reducing affinity to the mononuclear phagocyte system, reducing phagocytic phagocytosis or uptake by the reticulocyte system, are stable in the circulatory system, and have significantly increased half-lives, consistent with the results in Table 1. The difference between the group 2 and the group 3 is that the paclitaxel targeted liposome injected in the group 2 is not added with the star-shaped cholic acid functionalized polylactic acid, so that the sustained release effect of the preparation is obviously inferior to that of the group"3". Therefore, the paclitaxel targeted sustained-release liposome disclosed by the invention has an obvious sustained-release effect due to the introduction of the star-shaped cholic acid functionalized polylactic acid, and meanwhile, the white cell membrane covered on the surface of the paclitaxel targeted sustained-release liposome better avoids the recognition and phagocytosis of an endothelial reticulum system, and the synergistic effect of the two aspects ensures that the paclitaxel targeted sustained-release liposome has longer half-life period and average residence time, reduces the clearance rate, has a better long circulation effect, can reduce the administration times and enhance the treatment effect of the medicament.
4.4 drug concentration determination and targeting in tissues: placing each tissue sample to room temperature, adding into Tris-HCl (0.01mol/L) homogenate, extracting with ethyl acetate, centrifuging, collecting upper solution, vacuum rotary evaporating, dissolving residue with methanol-water (70:30), and determining paclitaxel content by high performance liquid chromatography, wherein the chromatographic conditions are as follows: the mobile phase is methanol-water (70: 30); the chromatographic column is elette C18(250mm × 4.6mm, 5 μm); the detection wavelength is 227 nm; the flow rate is 1.0 ml/min; the column temperature was 25 ℃; the amount of sample was 20. mu.L. The time-blood concentration of each tissue was inputted into the pharmacokinetic 3P87 program, and the area under the drug-time curve AUC (representing the cumulative total amount of paclitaxel in each tissue) and the tissue drug targeting efficiency Te (═ AUC) were calculated for each tissueTissue of/AUCBlood circulation) And relative tumor tissue uptake ratio AUCtumor/tissue(＝AUCTumor(s)/AUCTissue of) The results are detailed in Table 2.
TABLE 2 targeting efficiency and tumor tissue relative uptake ratio of paclitaxel in different formulations in tumor-bearing nude mice (example 1)
Tail vein injection of taxol (paclitaxel injection) for group "1" in the table; group 2' tail vein injection paclitaxel targeting liposome without star cholic acid functionalized polylactic acid; group 3 tail vein injection paclitaxel targeting slow release liposome.
Analyzing tissue distribution and targeting effect: after different formulations of paclitaxel are injected into tail vein, the difference of the target efficiency Te of the tumor tissue is obvious, the Te of the taxol (paclitaxel injection) is 0.71, which is obviously lower than that of two liposome formulations modified by white cell membranes (the Te is respectively 2.42 and 2.56). The results of relative uptake of tumor tissues in the table show that after two liposome formulations are injected by tail vein, the uptake of the tumor tissues relative to heart, lung, liver, spleen and kidney is obviously improved (P is less than 0.05) compared with that of taxol (paclitaxel injection), which indicates that liposome can obviously enhance the targeting ability of the drug after being modified by white cell membrane, so that the drug is more distributed in the tumor tissues, and other normal organs are relatively less distributed, so that the anti-tumor effect of the paclitaxel is enhanced, and the toxic and side effects are reduced. The group 2 is paclitaxel targeted liposome injected into tail vein without adding star-shaped cholic acid functionalized polylactic acid, the group 3 is paclitaxel targeted sustained-release liposome injected into tail vein, the tumor tissue targeting efficiency Te and the tumor tissue relative uptake ratio AUCtumor/tissue are both similar, which indicates that the addition of star-shaped cholic acid functionalized polylactic acid in liposome components has no influence on the targeting effect of the preparation. However, the tissue distribution test results show that the decrease speed of the paclitaxel targeted liposome without the addition of the star-shaped cholic acid functionalized polylactic acid is higher than that of the paclitaxel targeted sustained-release liposome containing the star-shaped cholic acid functionalized polylactic acid in blood and various tissues after the tail vein injection of a tumor-bearing nude mouse, and the concentration of paclitaxel in the group 2 and 6 hours is equivalent to that of the drug in the group 3 and 12 hours, which indicates that the paclitaxel targeted sustained-release liposome added with the star-shaped cholic acid functionalized polylactic acid disclosed by the invention not only has an excellent targeting effect, but also has a more excellent in vivo long-circulating effect.
Example 2
1. A preparation method of paclitaxel targeting sustained-release liposome modified by leucocyte membranes comprises the following steps:
the preparation method is the same as that of example 1, wherein the feeding amount of each component is as follows: 7.5mg of paclitaxel, 60mg of soybean phospholipid, 20mg of cholesterol, 7.5mg of star-shaped cholic acid functionalized polylactic acid and 5mg of leukocyte membrane.
2. Pharmaceutics characterization of paclitaxel targeted sustained-release liposome
(1) Particle size and polydispersity: the size is measured by a laser scattering particle size distribution instrument, and the measurement result is as follows: the average particle size is 87.5 nm; the polydispersity was 0.186.
(2) And surface appearance: the measurement method was the same as in example 1, and the measurement results were: the surface appearance is spherical, and the size distribution is relatively uniform.
(3) And the determination and the result of the encapsulation efficiency are as follows: the measurement method was the same as in example 1, and the measurement results were: the encapsulation efficiency was 85.7%.
3. In vitro cumulative drug release percentage test
The determination method and the calculation formula are the same as the example 1, and the paclitaxel targeted liposome (other components and processes are the same) without adding the star-shaped cholic acid functionalized polylactic acid is used as a reference preparation, and the cumulative drug release percentage is determined and calculated according to the same in vitro cumulative drug release percentage test method.
The result is shown in fig. 2 in detail, compared with the paclitaxel targeted liposome without the star-shaped cholic acid functionalized polylactic acid, the paclitaxel targeted sustained-release liposome prepared in this example releases the drug more slowly, the cumulative drug release percentages of 1, 2, 3, 6, 9 and 12h are 5.7, 14.2, 20.4, 40.7, 52.6 and 61.4 respectively, and the cumulative drug release percentages of the paclitaxel targeted liposome without the star-shaped cholic acid functionalized polylactic acid at the corresponding time points are 32.4, 47.5, 57.6, 69.7, 76.3 and 80.7 respectively, which indicates that the star-shaped cholic acid functionalized polylactic acid can play a role in delaying the drug release.
4. Pharmacokinetics and tissue distribution in vivo
4.1 establishing a nude mouse breast cancer animal model: the establishing method and the steps are the same as the embodiment 1.
4.2 dosing and sampling: the administration and sampling time points were the same as in example 1.
4.3 blood concentration determination and pharmacokinetic parameters: the plasma concentration measurement method and the pharmacokinetic procedure used were the same as in example 1, and the results are detailed in table 3.
TABLE 3 in vivo pharmacokinetic parameters of different formulations of paclitaxel (example 2)
Tail vein injection of taxol (paclitaxel injection) for group "1" in the table; group 2' tail vein injection paclitaxel targeting liposome without star cholic acid functionalized polylactic acid; group 3 tail vein injection paclitaxel targeting slow release liposome.
And (3) analyzing pharmacokinetic parameters: the results and analysis were in accordance with example 1.
4.4 drug concentration determination and targeting in tissues: the method of determining the concentration of drug in the tissue and the pharmacokinetic procedure and calculation formula used are the same as in example 1, and the results are detailed in Table 4.
TABLE 4 targeting efficiency and tumor tissue relative uptake ratio of paclitaxel in different formulations in tumor-bearing nude mice (example 2)
Tail vein injection of taxol (paclitaxel injection) for group "1" in the table; group 2' tail vein injection paclitaxel targeting liposome without star cholic acid functionalized polylactic acid; group 3 tail vein injection paclitaxel targeting slow release liposome.
Analyzing tissue distribution and targeting effect: the results and analysis were in accordance with example 1.
Example 3
1. A preparation method of paclitaxel targeting sustained-release liposome modified by leucocyte membranes comprises the following steps:
the preparation method is the same as that of example 1, wherein the feeding amount of each component is as follows: 10mg of paclitaxel, 55mg of soybean phospholipid, 24mg of cholesterol, 8mg of star-shaped cholic acid functionalized polylactic acid and 3mg of leukocyte membrane.
2. Pharmaceutics characterization of paclitaxel targeted sustained-release liposome
(1) Particle size and polydispersity: the size is measured by a laser scattering particle size distribution instrument. And (3) measuring results: the average particle size is 84.9 nm; polydispersity of 0.177;
(2) and surface appearance: the measurement method was the same as in example 1, and the measurement results were: the surface appearance is spherical, and the size distribution is relatively uniform.
(3) And the determination and the result of the encapsulation efficiency are as follows: the measurement method was the same as in example 1, and the measurement results were: the encapsulation efficiency was 84.6%.
3. In vitro cumulative drug release percentage test
The determination method and the calculation formula are the same as the example 1, and the paclitaxel targeted liposome (other components and processes are the same) without adding the star-shaped cholic acid functionalized polylactic acid is used as a reference preparation, and the cumulative drug release percentage is determined and calculated according to the same in vitro cumulative drug release percentage test method.
The result is shown in fig. 3 in detail, compared with the paclitaxel targeted liposome without the star-shaped cholic acid functionalized polylactic acid, the paclitaxel targeted sustained-release liposome prepared in this example releases the drug more slowly, the cumulative drug release percentages of 1, 2, 3, 6, 9 and 12h are 8.8, 1.78, 23.5, 45.3, 58.3 and 65.8 respectively, and the cumulative drug release percentages of the paclitaxel targeted liposome without the star-shaped cholic acid functionalized polylactic acid at the corresponding time points are 37.4, 50.6, 59.3, 70.1, 78.9 and 82.7 respectively, which indicates that the star-shaped cholic acid functionalized polylactic acid can be added to play a role in delaying the drug release.
4. Pharmacokinetics and tissue distribution in vivo
4.1 establishing a nude mouse breast cancer animal model: the establishing method and the steps are the same as the embodiment 1.
4.2 dosing and sampling: the administration method and the sampling time point were the same as those in example 1
4.3 blood concentration determination and pharmacokinetic parameters: the plasma concentration measurement method and the pharmacokinetic procedure used were the same as in example 1, and the results are detailed in table 5.
TABLE 5 in vivo pharmacokinetic parameters of different formulations of paclitaxel (example 3)
Tail vein injection of taxol (paclitaxel injection) for group "1" in the table; group 2' tail vein injection paclitaxel targeting liposome without star cholic acid functionalized polylactic acid; group 3 tail vein injection paclitaxel targeting slow release liposome.
And (3) analyzing pharmacokinetic parameters: the results and analysis were in accordance with example 1.
4.4 drug concentration determination and targeting in tissues: the method of measuring the concentration of the drug in the tissue and the pharmacokinetic procedure and the calculation formula used are the same as in example 1, and the results are detailed in Table 6.
TABLE 6 targeting efficiency and tumor tissue relative uptake ratio of paclitaxel in various formulations in tumor-bearing nude mice (example 3)
Tail vein injection of taxol (paclitaxel injection) for group "1" in the table; group 2' tail vein injection paclitaxel targeting liposome without star cholic acid functionalized polylactic acid; group 3 tail vein injection paclitaxel targeting slow release liposome.
Analyzing tissue distribution and targeting effect: the results and analysis were in accordance with example 1.
Claims (4)
1. A paclitaxel targeting sustained-release liposome modified by leukocyte membrane is characterized in that: the liposome consists of a drug-loaded sustained-release liposome consisting of paclitaxel, star-shaped cholic acid functionalized polylactic acid, soybean phospholipid and cholesterol and a leukocyte membrane modified on the surface of the drug-loaded sustained-release liposome.
2. The leukocyte membrane-modified paclitaxel targeted sustained-release liposome of claim 1, wherein: the liposome comprises the following components in percentage by mass: 5-10% of paclitaxel, 50-70% of soybean phospholipid, 15-25% of cholesterol, 6-12% of star-shaped cholic acid functionalized polylactic acid and 2-6% of leukocyte membrane.
3. A preparation method of paclitaxel targeting sustained-release liposome modified by leucocyte membranes is characterized in that: the method comprises the following steps:
(1) preparing paclitaxel sustained-release liposome: weighing paclitaxel, soybean phospholipid, cholesterol and star-shaped cholic acid functionalized polylactic acid, dissolving in chloroform, performing reduced pressure rotary evaporation at 35-40 ℃ to remove the solvent until a liposome film is formed, placing in a vacuum drier for 24 hours, adding a phosphate buffer solution, performing rotary hydration in a constant-temperature water bath at 40 ℃ for 1 hour, performing ultrasonic treatment on an ice bath probe for 4-6 min, processing by a micro-jet high-pressure homogenizer, and filtering by a microporous filter membrane to obtain a paclitaxel sustained-release liposome suspension;
(2) preparation of leukocyte membrane: separating leucocytes: selecting a rat, collecting whole blood through an eye socket, diluting the whole blood with an equivalent amount of EDTA-PBS buffer solution, and adding the diluted whole blood into a centrifugal tube for later use; mixing 9% of ficoll and 15.2% of diatrizoate meglumine, preparing a solution with the specific gravity of 1.114g/ml by using distilled water, adjusting the osmotic pressure to 540mOsm/L by using a 0.3% sodium chloride solution, adding the solution into the centrifugal tube along the tube wall, centrifuging for 30min by using the centrifugal force of 700 Xg, slowly absorbing the middle milky cloudy layer by using a liquid transfer gun, cleaning by using an EDTA-PBS buffer solution, centrifuging, and collecting white blood cells;
separating and purifying the leucocyte membrane: resuspending the collected leucocytes with leucocyte hypotonic lysate, homogenizing for 2min by a homogenizer in ice bath, performing hypotonic lysis for 24h at 4 ℃, centrifuging for 10min at a centrifugal force of 1200 Xg, discarding supernatant, centrifuging the collected leucocyte membranes for 30min at a centrifugal force of 24000 Xg at 4 ℃ by gradient sucrose solutions with three concentrations of 30%, 45% and 50%, respectively, collecting purified leucocyte membranes, and placing the purified leucocyte membranes in a refrigerator at 2-6 ℃ for later use;
(3) modification of paclitaxel sustained-release liposome by leukocyte membrane: and oscillating and mixing the prepared leucocyte membrane and liposome, incubating and fusing for 12h, centrifuging, washing by using a PBS buffer solution, then resuspending by using distilled water, performing ultrasonic treatment for 60s by using an ultrasonic instrument at the frequency of 35kHz and the power of 80W, passing through a microporous filter membrane, centrifuging at high speed, discarding supernatant, and freeze-drying to obtain the taxol targeted sustained-release liposome modified by the leucocyte membrane.
4. The method for preparing the paclitaxel targeted sustained-release liposome modified by the leukocyte membrane according to claim 3, wherein the method comprises the following steps: when the microjet high-pressure homogenizer in the step (1) is used for processing, the homogenizing pressure is 120MPa, the homogenizing temperature is 37 ℃, and the homogenizing times are 5 times.
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| "Lipid–polymer hybrid nanoparticles for synergistic drug delivery to overcome cancer drug resistance";Shao-Qi Zeng et al;《New Journal of Chemistry》;20171231;第41卷;第1518页摘要、第1521页示意图1、第1521页第2栏3.1、第1522页图3 * |
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